Proper control of the immune system is critical to human health. A hypersensitive immune system can lead to allergies and autoimmune disease, whereas impairment of the immune system leaves an individual susceptible to infections and even cancers. Dendritic cells (DCs) play an important role in ensuring proper regulation of the immune system. In response to pathogens, DCs rapidly alter their phenotype to limit the spread of infection, and initiate an adaptive immune response. DCs are also involved in preventing autoimmunity by maintaining self tolerance. It is now understood that DCs are not a homogeneous population of cells. They are comprised of functionally distinct subsets with different abilities to process antigens, respond to environmental stimuli, and engage distinct effector lymphocytes. DC subsets include the CD8+ DCs that are specialized in the cross-presentation of cell associated antigens to CD8+ T cells, and plasmacytoid DC (pDC), which help to establish an antiviral state through the rapid secretion of high amounts of interferon alpha. While we now appreciate that DCs are a heterogeneous population, little is known about the mechanisms that drive DC lineage commitment and differentiation, as well as the mechanisms that control functional specialization. Our preliminary profiling data and functional analysis have led us to hypothesize that specific microRNA (miRNA) play a key role in regulating DC identity. Indeed, when we knocked out a single miRNA specifically in DCs we observed a significant reduction in the number of pDCs, but not other subsets in mice, and the pDCs had a major reduction in their functional response to TLR stimulation. Here, we propose studies aimed at better understanding how this miRNA controls pDC development and function, including studies to identify the relevant targets of this miRNA (Aim 1). We will also investigate the role of two other miRNA that we identified through profiling studies to be differentially expressed between DC subsets (Aim 2). To do this, we will use a state-of-the-art miRNA decoy vector system that we generated, which enables stable inhibition of a miRNA in mouse DCs in vivo and in human DCs in culture. Finally, we will address a broader question of how miRNAs themselves are regulated in DCs using a novel, functional assay that we developed, which permits high resolution assessment of each miRNA's cellular activity (Aim 3). We have focused our efforts on eliciting the role of miRNAs in DCs because these small non-coding RNAs are known to play a role in controlling cell identity, and because the discovery of relevant miRNAs can be used to uncover other genes and pathways that are important to DC function through the identification of the miRNA's regulatory targets. Thus, our studies will not only provide new insight into the role of specific miRNAs, but they will also reveal other genes involved in the regulation of DCs. This, in turn, will help to find potential new causes of immune dysfunction, and to supply new strategies to enhance or subdue immune responses for the treatment of diseases, and in the aid of vaccine development.